TET2 regulates early and late transitions in exhausted CD8+ T cell differentiation and limits CAR T cell function

Abstract

CD8⁺ T cell exhaustion hampers control of cancer and chronic infections and limits chimeric antigen receptor (CAR) T cell efficacy. Targeting TET2 in CAR T cells provides therapeutic benefit; however, TET2's role in exhausted T cell (T_EX) development is unclear. In chronic lymphocytic choriomeningitis virus (LCMV) infection, TET2 drove conversion from stem cell-like T_EX progenitors toward terminally differentiated and effector (T_EFF)-like T_EX. TET2 also enforced a terminally differentiated state in the early bifurcation between T_EFF and T_EX, indicating broad roles for TET2 in acquisition of effector biology. To exploit the therapeutic potential of TET2, we developed clinically actionable TET2-targeted CAR T cells by disrupting TET2 via knock-in of a safety switch alongside CAR knock-in at the TRAC locus. TET2-targeted CAR T cells exhibited restrained terminal exhaustion in vitro and enhanced antitumor responses in vivo. Thus, TET2 regulates fate transitions in T_EX differentiation and can be targeted with a safety mechanism in CAR T cells for improved tumor control.

Fig. 1.. TET2-deleted CAR T cells adopt a central memory–like state following manufacturing and exhibit increased expansion and reduced IR expression under chronic stimulation across multiple CAR constructs.

(A) CAR T cell manufacturing and TET2 gene editing schematic. (B) Example plots and data after CAR T cell expansion (day 9), highlighting CCR7⁺ CD45RO⁺ central memory subset, n = 5. (C and D) Longitudinal oxygen consumption rate (OCR) (C), basal respiration, maximal respiration, and SRC (D) of TET2-disrupted cells at end of expansion (day 9) after oligomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and antimycin A/rotenone administration as indicated in (C), n = 5, run in triplicate. (E) Extracellular acidification rate (ECAR) of the same TET2-disrupted cells from (C) and (D). (F and G) Schematic of CD19.CD3ζ, CD19.BBζ, and CD19.CD28ζ CAR constructs ± TET2_KO (F) placed in a serial restimulation stress test (G). (H and I) Cumulative fold expansion of serially restimulated CAR T cells ± TET2_KO throughout (H) and at day 25 (I), n = 4. (J) Example plots and data of serially restimulated CAR T cells ± TET2_KO showing distribution of CCR7⁺ CD45RO⁺ central memory–associated markers in CD8⁺ CAR T cell populations after five stimulations, n = 4. (K) SPICE plots showing distribution of IR coexpression in serially restimulated CD8⁺ CAR T cells ± TET2_KO after five stimulations (chronic, day 25) with K562-CD19⁺ cells as depicted in (G), n = 4. (L) Example histograms (top) and data (bottom) of TCF1 gMFI in serially restimulated CD8⁺ CAR T cells ± TET2_KO after five stimulations, with indicated fold change, n = 4. (M) IL-2 and TNF production from supernatant collected 24 hours after fifth stimulation, n = 5. Data shown as means ± SEM [(C), (E), and (H)] or individual values [(B), (D), (I), (J), (L), and (M)] from independent donors. ns P > 0.05; *P < 0.05; **P < 0.01 by paired t test. Schematics [(A), (F), and (G)] created with BioRender.com.

Fig. 2.. TET2 mediates the transition out of the progenitor T_EX subset toward terminal exhaustion.

(A) Cotransfer experimental schematic. Inset plot shows initial P14 cotransfer. (B) Example plots and data for IR expression on TET2_KO P14 cells compared to WT P14 cells. (C and D) Example plots (C) and data (D) comparing IFN-γ and TNF expression following peptide restimulation for WT and TET2_KO P14 cells. (E and F) GSEA of terminal T_EX (E) and T_EX progenitor (F) signatures between WT and TET2_KO P14 cells at day 15 p.i. with LCMV clone 13 (Gene sets from GSE84105). (G) Example plots and data comparing TCF1⁺ T_EX progenitor and GZMB⁺ terminally differentiated T_EX frequencies within WT and TET2_KO P14 cells. (B and G) n = 9, spleen at day 30 p.i. with LCMV clone 13. Data for individual mice shown; representative of >4 independent experiments. (D) n = 5, spleen at day 37 p.i. with LCMV clone 13. Data for individual mice shown; representative of three independent experiments. [(B), (D), and (G)] ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by paired t test. Schematic (A) created with BioRender.com.

Fig. 3.. Loss of TET2 limits terminal differentiation of exhausted CD8 T cells.

(A) Experiment schematic for RNA-seq and ATAC-seq. WT and TET2_KO P14 cells were analyzed at day 15 p.i. with LCMV clone 13. (B) Sorting strategy for T_EX subsets for RNA and ATAC-seq. (C and D) PCA of RNA-seq (C) and ATAC-seq (D) data for WT and TET2_KO T_EX subsets. (E and F) Number of DACRs (E) or DEGs (F) for each pairwise comparison between WT and TET2_KO T_EX subsets {FDR < 0.05, with variable absolute log₂ fold changes [abs(log₂FC)] indicated}. (G) Volcano plot highlighting DEG in WT compared to TET2_KO LY108^neg T_EX. (H) Example plots and data comparing expression of PD1, CD39, and 2B4 on TET2_KO LY108^neg T_EX to WT LY108^neg T_EX. (I) Correlation plot of differential gene expression and peak accessibility in TET2_KO LY108^neg T_EX compared to WT LY108^neg T_EX with TOX labelled. (J) Example tracks showing accessibility at the Tox locus in LY108^neg T_EX. Differentially Accessible Peaks (DAPs) are indicated in orange. (K and L) Example plots (K) and data (L) comparing TOX expression in TET2_KO LY108^neg T_EX to WT LY108^neg T_EX. [(H) and (L)] n = 9, spleen at day 30 p.i. with LCMV clone 13. Data for individual mice shown; representative of three independent experiments. ns P > 0.05; ****P < 0.0001 by paired t test. Schematic (A) created with BioRender.com.

Fig. 4.. TET2 regulates early bifurcation of T_EX from T_EFF-like cells.

(A) Experiment schematic for analysis of TET2_KO P14 cells early (days 6 to 8) of LCMV clone 13 infection. (B) Example plots and data comparing frequency of early T_EFF-like cells (KLRG1⁺ PD1^low) within WT and TET2_KO P14 cells at day 8 p.i. (C) Frequencies of WT and TET2_KO KLRG1⁺ PD1^low T_EFF-like cells from total live splenocytes at day 8 p.i. (D) Example plots and data comparing expression of TCF1 and GZMB for WT and TET2_KO P14 cells. (E) Experiment schematic for rescue of TET2 function in TET2_KO P14 cells. (F and G) Example plots (F) and data (G) comparing frequencies of LY108⁺ T_EX and TIM3⁺ T_EX for WT and TET2_KO P14 cells with or without overexpression of the TET2 catalytic domain (TET2 CD versus MIGR1). (H and I) Example plots (H) and data (I) comparing frequencies of CD127⁺ T_MEM-like and KLRG1⁺ T_EFF-like cells for WT and TET2_KO P14 cells with or without overexpression of the TET2 catalytic domain (TET2 CD). (J) Model for TET2 role at major bifurcation events in chronic infection. [(B) to (D)] n = 5, spleen at day 8 p.i. with LCMV clone 13. Data for individual mice shown, representative of two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by paired t test. [(G) and (I)] n = 7, spleen at day 10 p.i. with LCMV clone 13. Data for individual mice shown, representative of two independent experiments. ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by multiple paired t test with Holm-Šídák posttest correction. Schematics [(A), (E), and (J)] created with BioRender.com.

Fig. 5.. Dual KI TET2-TRAC-CAR19 T cells have enhanced proliferation and maintain memory-associated marker expression.

(A) Left: Schematic of TRAC (top) and TET2 (bottom) loci alongside rAAV6 KI vectors. Right: Sanger sequencing electropherogram confirming integration of TRAC and TET2 KI constructs, underlined with dashed line. (B) Example plots of TET2 and TRAC-CAR19 single KI or dual TET2-TRAC-CAR19 KI T cells. (C) Example plots of CD3 loss detected by flow in TRAC-CAR19-KI T cells. (D and E) Schematic of in vitro ADCC assay (D) to deplete CRISPR-edited TET2-KI T cells. Example plots and data (E) of EGFR expression on TET2-KI T cells alone or in an NK cell coculture ± cetuximab incubation, gated on CD56⁻ populations, n = 4. (F) Cumulative fold expansion of TRAC-CAR19 and TET2-TRAC-CAR19 T cells during restimulation and at day 25, arrows represent addition of irradiated K562-CD19⁺ target cells, n = 5. (G) Proportions of CD4⁺ versus CD8⁺ T cells in TRAC-CAR19 and TET2-TRAC-CAR19 T cells after stimulation, n = 7. (H) Example plots showing distribution of central (CCR7⁺ CD45RO⁺) and effector (CCR7⁻ CD45RO⁺) memory-associated markers in CD8⁺ CAR T cell populations after restimulation, with summary after five stimulations, n = 5. (I) SPICE plot showing distribution of IR coexpression in CD8⁺ TRAC-CAR19 and TET2-TRAC-CAR19 T cells after 1 (acute) and 5 (chronic) stimulations, n = 6. (J) Data shown as means ± SEM [(F) and (G)] or individual values [(E) and (H)] from independent donors. ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 by paired t test. Schematics [(A and (D)] created with BioRender.com.

Fig. 6.. TET2 disruption limits terminal differentiation of TRAC-CAR19 T cells.

(A) Volcano plot showing differentially regulated genes in CD8⁺ TET2-TRAC-CAR19 and TRAC-CAR19 T cells after four stimulations; select markers highlighted. EGFR highlighted in red as TET2-KI construct positive control. Graph axes represent log₂ fold change and -log₁₀ P value, n = 3. (B) Box plots of individual gene log₁₀ Transcripts per Million (TPM) for TET2-TRAC-CAR19 and TRAC-CAR19 T cells from (A), n = 3. (C and D) GSEA for signatures of stem-cell central memory and central memory T cells (T_SCM and T_CM) and T_EX progenitors (gene sets from GSE147398) (C) and from human tumor infiltrating lymphocytes (TILs) [from (69)]. (D) Normalized enrichment score (NES) and FDR indicated in panel. (E and F) Example plots (E) and data (F) of CD8⁺ CAR T cell TCF1/Granzyme B subpopulation frequencies after four stimulations, n = 4. (G and H) Example plots (G) and data (H) of CD8⁺ CAR T cell TCF1/CD62L subpopulation frequencies after four stimulations, n = 4. (I and J) Number of CD8⁺ TCF1⁻ Granzyme B⁺, CD8⁺ TCF1⁺ Granzyme B⁻ (I) and CD8⁺ TCF1⁺ CD62L⁺ (J) CAR T cells from (F) and (H), n = 4. (K) Model for TET2 role during CAR T cell chronic stimulation. Data shown as means ± SEM (B) or individual values [(F) and (H) to (J)] from independent donors. ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 by paired t test. Schematic (K) created with BioRender.com.

Fig. 7.. TET2 KI enhances the antitumor activity of TRAC-CAR19 T cells in vivo.

(A) Overview of in vivo experimental design, n = 4 to 5 mice per experimental group; one experiment. (B) Longitudinal tumor burden of all experimental groups by bioluminescent imaging (BLI). (C to G) Tumor outgrowth for PBS only (C), T cell (D), TET2-KI T cell (E), TRAC-CAR19 T cell (F), and TET2-TRAC-CAR19 T cell (G) groups, with individual mice shown. (H) Longitudinal comparison of TRAC-CAR19 and TET2-TRAC-CAR19 T cell group tumor burden. (I) BLI comparison at days during and immediately after peak CAR T cell response in TRAC-CAR19 and TET2-TRAC-CAR19 groups, line at mean with SEM; ns P > 0.05; *P < 0.05; **P < 0.01 by multiple paired t tests with Holm-Šídák correction. (J) Overall group survival, **P < 0.01; ****P < 0.0001 by Mantel-Cox log-rank test. Data shown as means ± SEM (H) or individual values [(C) to (G) and I)] from each mouse. Schematic (A) created with BioRender.com.